Bailongjiang Watershed is an important area of water conservation in the upper reaches of the Yangtze River, with great significance for maintaining the ecological balance of the watershed and promoting regional sustainable development. Taking the Bailongjiang Watershed in Gansu as an example, we used the InVEST water conservation assessment model based on parameter localization to analyze the multi-scale spatiotemporal change of water conservation in the watershed from 1990 to 2016 and its influencing factors. The results showed that the average annual water conservation depth of the watershed was 47.50 mm, with a trend of decreasing first and then rising. In terms of spatial distribution, the high value areas were mainly concentrated in the steep overcast forest area of middle-high mountain in Wenxian, Diebu and Zhouqu (the conservation depth of the three counties, 2500-3500 m altitude section, shaded and semi-shaded slopes, slope section above 25° and the forest land was higher than the average conservation depth of the watershed). The low value areas were mainly distributed in the gentle apricus farming and pastoral areas of middle-low mountain in Tanchang and Wudu (the average conservation depth in the two counties, below 2500 m elevation, sunny slope and semi-sunny slope, below 25° slope, cropland and grassland was lower than the watershed). Under the climatic background, the spatial difference of land use/cover pattern driven by human activities was an important factor affecting the spatiotemporal change of water conservation. Returning cropland to forest is of great significance in slowing down the decline of water conservation depth in the context of climate warming.